Periodic Reporting for period 1 - PEPSA-MATE (Nanopeptides and Nanosaccharides for Advanced and Sustainable Materials) Reporting period: 2020-03-01 to 2023-02-28 Summary of the context and overall objectives of the project The purpose of PEPSA-MATE is to create a multidisciplinary team – including early career researchers – to derive novel products from polysaccharides (complex carbohydrates) and peptides nanoparticles using experimental and computer-based theoretical design approaches and innovative ultrasonic fabrication technologies. Being eco-friendly alternatives to conventional fossil fuel-derived materials, the engineered nanoparticles are used as drug-delivery systems and biodegradable plastic matrix. PEPSA-MATE outcomes expand knowledge base and research capability in Chemical biological Sciences and Technology by providing much-needed understanding and knowledge required to engineer new materials based on naturally sourced nanomaterials.PEPSA-MATE involves key participation of junior researchers providing strong support and training for the development of their careers. PEPSA-MATE develops intersectoral collaborations between a SME and academic partners to boost entrepreneurship creativity towards sustainable products and processes.PEPSA-MATE brings together interdisciplinary and complementary expertise in the field of nanomaterials, biopolymers, polysaccharides, cell biology, acoustics, and computational modelling. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far During the first reporting period, we have engineered a library of modified phytoglycogen nanoparticles, PG NP, with tunable structural and functional properties to be used as building blocks for the fabrication of new materials. First, we have developed a simple and cost-effective method to reduce PG NP size in a controlled manner ranging from 100 to 50 nm. We have tagged functional groups (amine, thiols, carboxyl groups) on the surface of glycogen NP. This allowed us to tune the surface charge and degradability against amylase of glycogen NP. The extent of chemical modification of PG NPs have been assessed by NMR spectroscopy. The size distribution and colloidal stability of modified PG NPs have been determined in aqueous suspension by dynamic light scattering, particle tracking measurements and GPC. The morphology and surface charge of the modified PG NPs have been investigated by electron microscopy, atomic force and super-resolution microscopy. Computer-based theoretical design of unmodified glycogen NP have been used to predict the chain mobility and flexibility, hydration of nanoparticles. The engineered glycogen nanoparticles were employed for the controlled and sustained delivery of therapeutic agents, i.e anticancer drugs and insulin. The results achieved so far on this research task have been published on high impact factor journals (Cavalieri et al. Nanoscale, 2022,14, 3452-3466. Cavalieri et al Advance Materials, 2023, 2210392, DOI: 10.1002/adma.202210392)Different methods for functionalising glycogen with thermoresponsive polymers have been explored. This has included polymerising functional monomers from the natural protein component of glycogen nanoparticles, in a “green” method of photopolymerisation in an aqueous-based system. The particles could be readily isolated and contained PNIPAM tethered chains. In addition, by coupling pre-formed temperature-responsive polymers directly on the glycogen nanoparticle via a grafting-to strategy we have produced glycogen-thermoresponsive polymers that will be tested as component in bioplastic films.A set of therapeutic peptides have been synthesized by stepwise solid-phase procedure and characterized by Mass Spectrometry. The spectroscopic and aggregation properties of the peptides were comprehensively investigated. Microscopy imaging experiments and Molecular Dynamics simulations were also carried out to investigate on the morphology of the aggregates and on the structure of the small peptide clusters nucleating the growth of large peptide aggregates. The amphiphilic peptides have been used as shell materials for the fabrication of oil-filled microcapsules. The results achieved so far have been published on good journals (Venanzi et al ChemPhysMater 2022, 1, 62-70. Venanzi et al Journal of Peptide Science, 2022, 28: e3356. Venanzi et al Nanomaterials 2022, 12(3), 466).Albumin protein microbubbles were first obtained by ultrasound assisted self-assembly of proteins at the oil-water interface. The microbubbles were characterized by optical microscopy. Then iron oxide nanoparticles with different size and shape were embedded into the albumin microbubbles to obtain a hybrid system endowed with magnetic properties and heat-responsive delivery properties.We have optimized the fabrication protocol of the MICROSPONGE (MSP) drug delivery platform, using two polysaccharides, i.e alginate and hyaluronic acid. The fabricated MSP were characterized by SEM and Dynamic Light Scattering. MSP were tested as platform for loading a release of therapeutic peptides synthesized by the partners. Finally, we have engineered glycogen-based nanocarriers for RNA delivery. To test the gene silencing ability of glycogen-RNA constructs we used prostate cancer cell line expressing luciferase (PC3-Luc). Glycogen-RNA nanoconstructs were prepared, characterized, and incubated with PC3-Luc cells to test their effect on cell luminescence expression. The results show a poor transfection efficiency of the constructs. Future studies will be focused on improving the efficiency of transfection of glycogen nanoparticles by changing their chemical and physical structure. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Innovations in functional nanomaterials have contributed significantly to the transformation of many industries, including manufacturing, healthcare, cosmetics, textiles, and electronics. The first-generation of nanoparticle is primarily based on non-degradable organic and inorganic materials. However, the potential impact of synthetic and non-degradable NPs on human health and ecosystems (e.g. microplastic pollution) is raising significant social and environmental concerns because of their potential long-term cytotoxicity and accumulation. Hence, sustainable, green, non-toxic, and degradable alternatives to first-generation man-made synthetic nanomaterials are urgently needed. Such nanomaterials need to be cost effective, simple, reproducible, and scalable, and can be exploited as building blocks for engineering biodegradable, non-toxic functional materials. PEPSA-MATE pursues a sustainable-oriented approach in turning nanoparticles naturally sourced on a large-scale from sweet corn, and custom-designed peptides into products using innovative technologies. IMPACT: During the secondments ESR involved in PEPSA-MATE developed communication and independent thinking and professional maturity. The partners produced 12 joint OA publications of high scientific value. The partners applied for new joint research grants (Horizon Europe RIA/IA) and have organized and international conference on Peptide Materials 2023, 26-29 October, Sorrento Italy for promoting the results of the ongoing research activities. PEPSA-MATE partners engaged numerous companies, stakeholders, and users to communicate the research outputs and identify possible opportunities to transfer our technologies. The coordinator set up a webpage to communicate and disseminate the results generated in the 1st RP. The partners have implemented the outreach plan to target multiple audiences.